Hrp conjugated donkey anti rabbit igg antibody

After 72 h of incubation with or without canagliflozin in the presence of 5.5 mM or 25 mM glucose, MCs were collected and sonicated in 400 µl of solubilizing solution containing 9 M urea, 2% Triton X-100, and 1% dithiothreitol. Then, 100 µl of 10% lithium dodecyl sulfate was added to the solubilized membrane fraction and sonicated again. Subsequently, proteins were separated discontinuously on 7.5% sodium dodecyl sulfate polyacrylamide gels and transferred to PVDF membranes (Bio-Rad, Hercules, CA, USA). After blocking nonspecific binding, the membranes were incubated overnight at 4 °C with rabbit anti-NOX4 (1:1000; Abcam), anti-PKC (1:250; Abcam), anti-pan phospho-PKC (1:1000; Cell Signaling Technology), anti-SGLT2 (1:500; Abcam), anti-TGF-β (1:500; Cell Signaling Technology), or anti-fibronectin (1:5000; Abcam) antibodies, followed by HRP-conjugated donkey anti-rabbit IgG antibody (1:10,000; Amersham) as a secondary antibody. The membranes were also incubated with goat anti-β-actin HRP-conjugated antibody (1:5000; Santa Cruz) to ensure equal protein loading of the lanes. We used the ECL Plus system (Amersham) to detect antibody binding.

E. coli isogenic strain MK602 (leu⁺mutT) was provided by Dr. M. Sekiguchi (2 (link)), and BL21 (DE3) was purchased from Novagen (Madison, WI). Plasmids pCR®II and pET28a (+) were obtained from Invitrogen (Carlsbad, CA) and Novagen (Madison, WI), respectively. Mouse monoclonal anti-histidine antibody was from Clontech (Palo Alto, CA) and horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG antibody was from Santa Cruz Biotechnology (Santa Cruz, CA). HRP-conjugated donkey anti-rabbit IgG antibody, ECL kit, and Hyperfilm-MP film were obtained from Amersham Pharmacia Biotech (Buckinghamshire, England).